CYTOLYSIN GENE AND GENE PRODUCT

Abstract: A Salmonella gene, encoding a cytolysin, has been identified by screening for hemolysis on blood agar. The gene (slyA) is present in every strain of Salmonella examined in Shigella, and enteroinvasive Escherichia coli (EIEC) but not in other enterobacteriaceae. It is encoded near 28.5 minutes on the chromosome. A SlyA (salmolysin) has hemolytic and cytolytic activity and has a molecular weight predicted by the DNA sequence.

Description: CYTOLYSINGENEANDGENEPRODUCT Background of the Invention Many Gram negative and Gram positive pathogenic microorganisms produce toxins or hemolysins that can lyse eukaryotic cells and contribute to their pathogenicity. S.A. Mims (1982), The Pathogenesis of Infectious Disease (Academic Press). The most intensively studied exotoxin produced by Gram negative pathogens are members of the RTX family and are reviewed in A. Ludwig and W. Goebel (1991), Genetic determi¬ nants of cytolytic toxins from gram-negative bacteria; in J.E. Alouf and J.H. Freev (eds.), Sourcebook of bacterial proteins, pp. 117-146, Academic Press, London; and in R.A. Welch (1991 ), Mol. Micro. 5, 521-528, and V. Braun et al. (1991 ), Critical Reviews in Microbiology 18, 115-158. The HlyA hemolysin present in strains of uropathogenic E. Coli is the best studied of these. HlyA increases E. Coli virulence in a rodent model of peritonitis. R.A. Welch et al. (1981), Nature 294, 665-667. Although the precise mechanism by which this occurs is unclear, it is suggested that these toxins are involved in attenuating host phagocytic cell function. Welch et al., supra; S. Bhakdi et al. (1986), Infect. Immun. 52, 63-69; S. Bhakdi et al. (1990), J. Clin. Inv. 85, 1746-1753; S.J. Cavalleri et al. (1984), Infect. Immun. 37, 966-974; and B. Konig et al. (1986), Infect. Immun. 54, 886-892. Several facultative intracellular pathogens, including rickettsiae, shigellae, Trypanosoma cruzi, and Listeria monocytogenes share a common trait, i.e., to allow escape from the phagocytic vesicle of professional phagocytic cells. J.W. Moulder (1991), cro. Rev. 55, 143- 190; J. Turco et al. (1991 ), Infect. Immun. 59, 1647-1655; B.B. Finlay et al. (1989), Micro. Rev. 53, 210-230; and N.W. Andrews et al. (1990), Cell. 61, 1277-1287. A defined role in virulence is difficult to assign to most toxins, in part because of the lack of convenient animal models for many orga¬ nisms. One exception is the listeriolysin made by Listeria monocytogenes. The toxin dissolves the phagocytic membrane, allowing the bacteria to escape into the cytoplasm; hemolysin negative mutants are avirulent. J.L. Gaillard et al. (1987), Infect. Immun. 55, 1641-1646; S. Kathariou et al. (1987), J. Bad 169, 1291-1297; and D. Portnoy et al. (1989), Infect. Immun. 57, 477-486. The infected cell remains intact, and the bacteria are protected from the immune defenses of the host. CM. Hage-Chahine et al. (1992), Infect. Immun. 60, 1415-1421. Transfer of the gene-encoding listeriolysin to the non-pathogenic bacterium Bacillus subtilis allows it to also escape the phagosome, suggesting that listeriolysin is both neces¬ sary and sufficient for this event. J. Bielecki et al. (1990), Nature 345, 175-176. It was observed that under the correct conditions, colonies of Salmonella were slightly hemolytic when cultured on blood agar plates. The segment of DNA encoding slyA was isolated from bacterial colonies expressing salmolysin by performing a series of deletion maps, carefully screening for hemolytic activity. The segment of DNA encoding the hemolytic activity was thus narrowed down to a specific region of the chromosome. Summary of the Invention The terms "slyA gene" and "Salmolysin" as used herein refer to genes and their protein products substantially homologous to, and having substantially the same biological activity as, the protein coded by the nucleotide sequences depicted in Figure 1 [Sequ.ID.No. 1]. This definition is intended to encompass allelic variations in the slyA gene sequence. This definition further encompasses the disclosed sequences of Figure 1 , [Sequ.ID.No. 1], [Sequ.ID.No. 3], [Sequ.ID.No. 4], and [Sequ.ID.No. 5], as well as their respective complements. All references to the slyA gene, and to nucleotide sequences thereof are intended to encompass such allelic variations, both naturally-occurring and man-made, as well as the respective complements. Similarly all references to amino acid sequences of Figure 1 and [Sequ.lD. No. 2] are intended to encompass such allelic variations, both naturally-occurring and man-made. Cloned genes of the present invention may code for slyA genes of any species of origin, including but not limited to, Salmonella, Shigella, Escherichia, and other bacterial strains, most preferably of Salmonella origin. The present invention relates to a Salmonella gene (slyA) encoding a cytolysin, or salmolysin, having hemolytic and/or cytolytic activity. The invention also relates to genes encoding a polypeptide having the biological activity of a salmolysin. These genes may be either naturally- occurring, chimeric, gene fragments thereof, or genetically-engineered, e.g., by site-directed mutagenesis, by chemical mutagens, radiation, transposons, or oligonucleotides. DNA and/or RNA sequences which hybridize to the slyA gene or a derivative thereof (e.g., an oligonucleotide of the slyA gene or their complement sequences) are also in the scope of the invention. Hybridizing sequences may be selected by their ability to hybridize specifically (e.g., selectively) the salmolysin gene or a fragment thereof under conditions which are generally suitable for selecting DNA sequences (e.g., see Molecular Cloning, Sambrook et al., 2nd Ed., 1989, especially 11.45-11.61 ). The source of DNA sequences capable of hybridizing with the slyA gene or gene fragment may be other bacterial strains, such as disclosed below in Figure 2, or, e.g., bacillus, aspergillus, amoeba, fungi, yeast, slime mold, insect, bird, mammals, and generally other living organisms and viruses. Although the Salmonella gene isolated herein is used as a representative example, it is recognized that other genes having cytolysin and/or hemolysin activity may be analogously isolated and used as described below. Thus, an aspect of the present invention is a salmolysin gene or salmolysin from any naturally-occurring source. The present invention also relates to oligonucleotides of the slyA gene or a gene which hybridizes to the slyA gene. The oligonucleotides may contain coding sequences for the slyA gene product or they may contain sequences from non-coding regions of the slyA gene, including, e.g., enhancer, promoter, transcription terminator, and other 5' and 3' sequences from the slyA gene product. The oligonucleotides may be employed, e.g., in the polymerase chain reaction, as probes, and in sequencing (e.g., see Molecular Cloning, Sambrook et al., 2nd Ed. 1989). The oligonucleotides may be used to amplify specific regions of the slyA gene by polymerase chain reaction for the purpose of identifying the presence of bacteria possessing the gene. For example, the presence in food stuffs, e.g. poultry, eggs, egg products, and meat, or in body fluids, e.g., blood, serum, saliva, feces, and urine, or in tissues, of strains having the slyA gene may be detected with a slyA probe by hybridization followed by the polymerase chain reaction. Since strains having the slyA gene may be pathogenic to living organisms, the ability to specifically and quickly detect their presence is especially advantageous for diagnostic and treat¬ ment purposes. For example, Salmonella, Shigella and EEC can cause an inflammatory diarrhea. Salmonella more often causes a disseminated infection, while Shigella and EIEC remain within the colonic epithelium, rarely spreading past the lamina propria to produce a disseminated infection. A slyA gene, gene fragment, or oligonucleotide thereof may be used as a hybridization or amplification probe as conventional in the art, inclu¬ ding, in solution, on solid or semi-solid supports, e.g., agarose (Northern or Southern) or polyacrylamide gels, nitrocellulose, nylon, and DEAE- cellulose membranes, microtiter wells, on tissue sections, whole tissue mounts, salivary and chromosome squashes, cells, or by PCR. The methods for hybridization may be performed, e.g., as in Molecular Cloning, Sambrook et al., 2nd Ed., 1989. For the purposes of performing PCR, oligonucleotides of a desired length may be selected which are specific for regions of the slyA gene for the purpose of amplifying and detecting the presence of a gene fragment in the sample DNA. Thus, an aspect of the present invention is also a method of detecting the presence of the slyA gene and e.g. thus the corresponding organisms, preferably Salmonella, Shigella, and enteroinvasive E. Coli (EIEC), having the slyA or a related gene; e.g., a gene that cross-hybri¬ dizes with the slyA gene. In one embodiment, a nucleic acid specific for the slyA gene is hybridized with a sample containing nucleic acid in which the presence of the organism is to be detected and the presence or absence of binding is determined. The sample may be a food stuff, e.g. poultry, eggs, egg products, and meat, or blood, urine, feces or other body fluids and tissues which contain the organism having the slyA gene. The sample may also be prepared from the latter sources by standard methods of isolating or nucleic acid for test purposes. The method may involve hybridizing and detecting on solid support, e.g., gels and papers, or by PCR. Hybridization may be detected routinely, e.g., by radioactive or enzyme-conjugated or fluorophore-conjugated probes. This invention is also a method of detecting the presence of the slyA gene, comprising hybridizing two different oligonucleotides according to claim 5 with a nucleic acid containing sample in which the presence of the slyA is to be detected, the conditions of such hybridizing being effective for said specificity of the slyA gene; performing the polymerase chain reaction on the hybridized sample; and detecting the presence or absence of a DNA fragment formed by the polymerase chain reaction. By "conditions of such hybridizing being effective for said specificity of the slyA gene" is meant that hybridization conditions are chosen so that the probe selective¬ ly detects a slyA gene in the nucleic acid sample. The selection of such conditions is routinely determined. DNA encoding the salmolysin slyA gene may be obtained by chemical synthesis, by screening cDNA or genomic libraries prepared from mRNA or genomic DNA, respectively, from appropriate cells or cell line cultures, or by a combination of these procedures, using methods well known in the art. Screening of mRNA or genomic DNA may be carried out with oligonucleotide probes generated from the slyA gene sequence provided herein. Probes may be labeled with a detectable group such as a fluorescent group, a radionuclide, or a chemiluminescent group, or with an enzyme label in accordance with known procedures and used in conven¬ tional hybridization assays. Alternatively slyA gene sequences may be obtained by in vitro amplification, for example, the polymerase chain reaction (PCR), with oligonucleotide primers as provided herein (see US Patents 4,683, 195 and 4,683,202 to Mullis). The invention also relates to 5 * sequences upstream and 3' sequences downstream from the slyA gene. For example, such sequences may be used as heterologous promoters or transcription terminators to control the expression of heterologous genes. Such uses may be accomplished conventionally, (e.g., see Methods in Enzvmology, Volume 185, Gene Expression Technology, Ed. by D.V. Goeddel, 1990), but substituting the novel sequences of the slyA gene. The present invention also relates to a slyA gene product, such as salmolysin. The gene product may be prepared according to methods which are conventional in the art, including by transforming microorga¬ nisms, e.g., E coli, bacillus, yeast, and aspergillus, with an expression vector comprising the slyA gene. For example, the slyA gene may be placed under control of the T7 promoter in an expression vector. Upon transformation into E coli, large quantities of the protein are produced (see Example 3, below). A salmolysin may also be purified from a natural source by chromatography, e.g., by anion exchange. Thus, another aspect of the invention is an isolated salmolysin. By "isolated", it is meant that the salmolysin is in a form which is not found in nature, preferably at least of a purity where it could be used to elicit antibodies or serve as a vaccine. Another aspect of the present invention is peptides of a slyA gene product. The peptides may be employed as immunogens for vaccinating organisms susceptible to infection by, e.g., Salmonella, Shigella, and enteroinvasive Echerichia coli (EIEC). In addition, the peptides may be used to elicit antibodies to a slyA gene product, e.g., for purposes of detection or in immunoassays. The peptides may also be useful for blocking antibody binding to determine antibody specificity in, e.g., immun¬ oassays and Western blots. These and other immunological techniques are described in e.g., Methods of Enzymology, Volumn 73, ed. by J. Langone and H. Van Vunakis, 1981. Thus, an aspect of the invention is an isolated peptide having a sequence of a salmolysin, e.g, according to Figure 1 [Sequ.ID.No. 2], wherein the peptide possesses an activity. The activity may include, e.g., the ability to elicit an immune response, inhibit salmolysin activity or expression, and encode a salmolysin activity. Antibodies that react with a slyA gene product, or a product of gene capable of hybridizing with the slyA gene, or peptides thereof also relate to the present invention. The antibodies may be naturally-occurring when isolated from an organism which is infected with an organism expressing a slyA or related gene. The antibodies of the present may also be raised to a salmolysin or a peptide thereof. Antibodies in the scope of the invention are monoclonal, polyclonal, fragments thereof, e.g., Fab, (Fab)2, single- chain, chimeric , and genetically-engineered antibodies or peptides capable of reacting with a salmolysin or related gene product. See, e.g., U.S. Patent Nos. 4,816,567, 5,091 ,513, 4,444,878, 4,642,334 and 4,978,745. The peptides and antibodies of this invention may be used in the preparation of diagnostic tests, such as immunoassays. Such diagnostic techniques include, for example, enzyme immune assay (EIA), enzyme multiplied immunoassay technique (EMIT), enzyme-linked immunosorbent assay (ELISA), radioimmune assay (RIA), fluorescence immune assay, either single or double antibody techniques, and other techniques where either the peptides or antibodies of this invention are labeled with some detectable tag. See generally, Enzyme Immunoassay, by Maggio, CRC Press (1981 ). A method of detecting salmolysin and/or organisms expressing salmolysin is therefore another aspect of the invention. For example, the method may comprise a method of immunologically detecting salmolysin comprising, reacting a sample in which the presence or absence of salmo¬ lysin is to be detected with an antibody specific for the slyA gene product, and detecting the presence or absence of antibody binding. A sample may be a body fluid, urine, feces, or a tissue which has been treated so that it may be used in immunological detection. Another aspect of the invention is an immunoassay for the purpose of detecting slyA in gut, comprising reacting an antibody specific for the slyA gene product and a sample comprising the slyA gene product, and detecting binding of the antibody to the slyA gene product in the sample. The peptides and antibodies of the present invention may be used as therapeutic formulations, e.g., as vaccines for providing protection against organisms which produce the slyA gene. They may be administered in any conventional dosage formulation, optionally with physiologically acceptable carriers which are generally used in the art. The present invention is also a kit comprising oligonucleotides which are specific for the salmolysin gene. For example, oligonucleotides, such as oligo 1 [Sequ.ID.No. 3], oligo 2 [Sequ.ID.No. 4], or oligo 3 [Sequ.ID.No. 5], according to Figure 1 , or their complement thereof, may comprise a kit for the purposes of PCR. The kit may be used to detect the presence of organisms which express a slyA gene (for diagnostic purposes.) The kit may include oligonucleotides, buffers, and other reagents used to detect the presence of a nucleic sequence which hybridizes to the oligonucleo¬ tides. The oligonucleotides may contain conjugated fluorophores or enzymes, in which case the kit would include the reagents necessary to detect the fluorophores or enzymes. The kit may also be used in conjunc¬ tion with a polymerase chain reaction, comprising the reagents to carry out the reaction, including, e.g., oligonucleotide primers to a slyA gene, buffer, DNA polymerase and dNTPs. (See Sambrook et al. supra.) A. Strains and Media: Wild-type ATCC strain Salmonella typhimurium 14028s was used in all virulence studies. Clinical isolates of Salmonella were obtained from the State of California Health Laboratory or the County of San Diego Health Laboratory. Bacteria were cultivated in Luria Bertani medium. Blood agar plates were made in trypticase soy agar (TSA Difco) containing 4% defibrinated sheep red blood cells (Colorado Serum, Denver). Clinical isolates obtained for homology studies included: Yersinia sp., Legionella, Chlamydia, Pasteurella, Acinetobacter, Haemophilus, Proteus, Klebsiella, Neisseria sp., Citrobacter, Campylobacter, Franciscella, Brucella, Listeria, Serupia sp., E. Coli K-12, and Aeromonas. B. Molecular Techniques: A Sau3A partial digest cosmid library of Salmonella typhimurium 14028s was constructed in the vector pl_ARF2 [A.M. Friedman et al. (1982), Gene. 18, 289-296] and packaged into lambda particles (Stratagene, La Jolla, California). E. Coli LE392 was used as the host for the cosmid library and was plated onto TSA blood plates containing 20 μg/ml tetracycline and incubated for 36 hours at 37°C to detect zones of hemolysis. A strongly hemolytic colony was chosen for further characterization. Subcloning, sequencing, and Southern analysis were performed by standard techniques [J. Sambrook et al. (1989), Molecular Cloning: A Laboratory Manual, Second Edition (Cold Spring Harbor: Cold Spring Laboratory Press)] in either M13 mp19 or pKS (Stratagene, LaJolla, California) using the Sequenase kit (United States Biochemicals). Sequence manipulation and data base searches were performed using IBI MacVector and GenBank. C. Chromosomal Location of the sly Gene and Southern Hybridizations: A series of "locked-in" Mu -P22 phage insertions at 3- minute overlapping intervals around the Salmonella typhimurium chromo- some has been created. N.R. Benson et al. (1992), J. Bad. 175, 1673- 1681 , and P. Youderian et al. (1988), Genetics 118, 581-592. Each strain packages up to 120 kb (about 3 minutes) of adjacent chromosome when induced with mitomycin C at 1 μg/ml. Phage particles were pelleted from the resulting lysates, and DNA was prepared by phenol extraction. One μ g of DNA from each region of the chromosome was immobilized on Nytran using a slot blot apparatus (Schleicher and Schuell) and hybridized with the salmolysin structural PCR amplified gene. All Southern hybridizations were done in 6x SSPE, 10x Denhardts, 0.5% SDS at 65°C and filters washed in 0.1 x SSPE at 65°C. Hybridization, PCR, and other general methods and solutions which are useful in the present invention are described in Sambrook et al., supra. D. Disruption of the sly Gene: The sly gene was disrupted by homologous recombination insertion of a suicide vector derived from the RK2 replicon, pFR10. R.C. Roberts et al. (1990), J. Bact. 172, 6204-6216. The essential replication gene, trfA, was deleted from this vector and replaced with an internal Ssp1 fragment of slyA. The suicide vector-sly fragment, pSL2145, was maintained in E. Co// S17-1 [R. Simon et al. (1983), Molecular Genetics of Plate-Microbe I Interactions, A. Puhlar, ed. (St. Paul Minnesota: APS Press), pp. 98-106] and transferred to wild-type Salmonella by conjugation. Transconjugates were selected on Brilliant green agar containing sulfadiazine (80 μg/ml) and penicillin (300 μg/ml). Because Salmonella recipient cells lack triA, penicillin resistance can only be stably maintained in recipients by homologous recombination between the internal sly fragment on the suicide plasmid and the sly gene on the chromosome, resulting in sly disruption. Chromosomal DNA was prepared from several penicillin-resistant colonies, restricted with Pstl, EcoRV, Clal, and EcoRV/Cla\, transferred to a membrane, and probed with the 32 P- labelled PCR amplified slyA gene to confirm the interruption of slyA. PCR may be performed as described in Innis et al. (1990), PCR Protocol: A Guide to Methods and Applications (Academic Press). All penicillin- resistance colonies examined showed disruption of the slyA gene. E. Virulence Determination of sly Mutants: Overnight cultures of 14028s (wild type) and SL2161 (slyA-) were grown in LB and LB contai¬ ning penicillin (200 μg/ml), respectively, and given orally to mice in 200 μl doses with a feeding cannula. Intraperitoneal and intravenous inocula¬ tions were given with bacteria washed and diluted in phosphate-buffered saline. LD50 values were determined over a four-week period. Course of infection studies were performed by homogenizing tissues in a Stomacher (Tekmar) from infected mice in sterile water, diluting the homogenates in phosphate-buffered saline, and plating or LB agar to determine the number of bacteria per organ. F. Expression and Partial Purification of a Salmolysin: Methods for the expression of cloned genes in bacteria are well known. To obtain high level expression of a cloned gene in a prokaryotic system, it is often essential to construct expression vectors which contain, at the minimum, a strong promoter to direct mRNA transcription termination. Examples of regulatory regions suitable for this purpose are the promoter and operator region of the E. coli β-galactosidase gene, the E. coli tryptophan biosyn- thetic pathway, a T7 or other RNA polymerase promoter or the leftward promoter from the phage lambda. The inclusion of selection markers in DNA vectors transformed in E. coli are useful. Examples of such markers include the genes specifying resistance to ampicillin, tetracycline, or chlor- amphenicol. See Sambrook, supra, for details concerning selection markers and promoters for use in E. coli. The oligonucleotide primers shown in Figure 1 [Sequ.lD.No ' s. 3, 4, and 5] were used to amplify a 515 bp fragment that contained an Nde\ site modified at the start codon and cloned into pRK172 [AH. Rosenberg et al. (1987), Gene. 56, 125-136] at the Λ/cfel site, placing salmolysin expression under the control of the T7 RNA polymerase promoter. The construct, pSL2042, was transformed into E. Coli B121 DE3 [F.W. Studier et al. (1990), Methods in Enzymology (Academic Press, San Diego), Vol. 185, pp. 6-89] and expression of salmolysin was induced by 1 mM IPTG (iso- propylbetagalactoside) in the presence of 200 μg/ml rifamplcin and 50 μCI/ ml 35 S-methionine. Proteins were separated by 12% SDS-PAGE, gel stained with Coomassie Brilliant Blue, dried, and exposed to X-ray film. Salmolysin was released from E. Coli containing pSL 1117 by the osmotic shock procedure of Neu and Heppel. H.C. Neu et al. (1965), J. Biol. Chem. 240, 3685-3692. Proteins were separated by FPLC monoQ anion exchange (Pharmacia) with a 0-1 M NaCI gradient, 50 mM Tris-HCI, pH 7.5 and 2 μg/ml of PMSF. Column fractions were assayed for hemolytic activity by adding 50 μl of each fraction to 700 μl of 10% defibrinated, washed sheep erythrocytes and incubated at 37°C for 1 hour. The amount of released hemoglobin was determined by measuring the optical density of the supernatant at 595 nm. Salmolysin eluted at 150 mM NaCI. Salmo- lysin could be further purified using preparative isoelectric focusing with a Fletofor (BioRad): all hemolytic activity focused at a pi of 5.5. A hemolytic unit of salmolysin was defined as the amount of partially purified salmo ¬ lysin required to lyse 50% of a suspension of 10% erythrocytes at 37°C in 1 hour. One hemolytic unit of salmolysin was heated to 65°C for 15 minutes, then assayed for activity. EDTA (50 mM final) was added to one hemolytic unit of salmolysin and treated as above. Phospholipase D and cholesterol oxidase activities were assayed by the method described in J.G. Songer et al. (1990), Infect. Immun. 58, 131-136. G. Macrophage Preparation and Infection: Peritoneal macro- phages were elicited from Balb/c mice by intrape toneal injection of proteous peptone, cultured, and infected with wild type and the salmolysin mutant as described in N.A. Buchmeier et al. (1989), Infect. Immun. 57, 1- 7. H. General Methods of Molecular Cloning and Genetic Engineering: This invention embraces molecular genetic manipulations that can be achieved in a variety of known ways. The recombinant cells, plasmids, and DNA sequences of the present invention provide a means to produce pharmaceutically useful compounds wherein the compound is a salmolysin. Generally, the definitions of nomenclature and descriptions of general laboratory procedures used in this application can be found in J. Sambrook et al., Molecular Cloning, A Laboratory Manual, (1989) Cold Spring Harbor Laboratory, Cold Spring Harbor, New York. The manual is hereinafter referred to as Sambrook and is hereby incorporated by reference. In addition, Ausubel et al., eds., Current Protocols in Molecular Biology, (1987 and periodic updates), Greene Publishing Associates, Wiley-lnterscience, New York, discloses methods useful in the present application. All enzymes are used according to the manufacturer's instructions. Oligonucleotides that are not commercially available can be chemi¬ cally synthesized according to the solid phase phosphoramidite triester method first described by S.L. Beaucage and M.H. Caruthers, (1981) Tetrahedron Letts., 22(20): 1859-1862 using an automated synthesizer, as described in D.R. Needham-VanDevanter et al., (1984) Nucleic Acids Res.. 12:6159-6168. Purification of oligonucleotides is by either native acrylamide gel electrophoresis or by anion-exchange HPLC as described in Pearson, J.D., and Regnier, F.E. (1983) J. Chrom., 255:137-149. Nucleotide sizes are given in either kilobases (kb) or base pairs (bp). These are estimates derived from agarose or acrylamide gel electrophoresis or from published DNA sequences. The sequence of the cloned genes and synthetic oligonucleotides can be verified using the chemical degradation method of Maxam, A.M., et al., (1980) Methods in Enzvmology. 65:499-560. or similar methods. The sequence can be confirmed after the assembly of the oligonucleotide frag¬ ments into the double-stranded DNA sequence using the method of Maxam and Gilbert, supra, or the chain termination method for sequencing double-stranded templates of Wallace, R.B., et al., (1981 ) Gene, 16:21-26. Southern Blot hybridization techniques were carried out according to Southern et al., (1975) J. Mol. Bio!.. 98:503. Embodiments of this invention often involve the creation of novel peptides and genes by in vitro mutagenesis. Target genes are isolated in intermediate vectors and cloned for amplification in prokaryotes such as E coli, Bacillus, or Streptomyces. Most preferred is E. coli because that organism is easy to culture and more fully understood than other species of prokaryotes. The Sambrook manual contains methodology, e.g., to conduct cloning and genetic engineering methods, E. coli strains may be grown in Luria broth (LB) with glucose, or M9 medium supplemented with glucose and acid-hydrolyzed casein amino acids. Strains with resistance to antibiotics were maintained at the drug concentrations described in Sambrook. Transformations may be performed according to the method described by Morrison, D.A. (1977) J. Bact.. 132:349-351 or by Clark- Curtiss, J.E., and Curtiss, R. (1983) Methods in Enzvmoloαv. 101 :347-362, Eds. R. Wu et al., Academic Press, New York. I. Peptides and Antibodies. The peptides can be prepared by conventional processes for synthesizing peptides; more specifically, using processes as described in Schroder and Lubke, The Peptides, Vol. 1 , published by Academic Press, New York (1966) or Izumiya, et al., Synthesis of Peptides. published by Maruzen Publishing Co., Ltd., (1975). For example, an azide process, an acid chloride process, a symmetric anhydride process, a mixed anhydride process, a DCC process, an active ester process (for example: p-nitrophenyl ester, N-hydroxysuccinimide ester, or cyanomethyl ester), a carbodiimidazole process, an oxidative- reductive process, or a DCC/additive process can be used. Solid phase and solution phase syntheses are both applicable to the foregoing processes. The peptides of the present invention are suitably prepared in accordance with the above processes as typically employed in peptide synthesis, generally either by a so-called stepwise process which compri¬ ses condensing an amino acid to the terminal amino acid, one by one in sequence, or by coupling peptide fragments to the terminal amino acid. Amino groups that are not being used in the coupling reaction and other reactive groups must be protected to prevent coupling at an incorrect location. Procedures of protecting such groups and of removing the protective groups are known in the art. In case that a solid phase synthesis is adopted, the C-terminal amino acid is bound to an insoluble carrier or support through its carboxyl group. The insoluble carrier is not particularly limited as long as it has a binding capability to a reactive carboxyl group. Examples of such insoluble carriers include halomethyl resins of polystyrene, such as chloromethyl resins, phenol resins, tert-alkyloxycarbonyl-hydrazidated resins, and the like. In general, antibodies may be obtained by injecting the desired immunogen or antigen into a wide variety of vertebrates, in accordance with conventional techniques. Suitable vertebrates include mice, rats, rabbits, sheep, and goats, with mice being preferred. Usually, the animals are bled periodically with the successive bleeds having improved titer and specificity. The antigens may be injected intramuscularly, intraperitoneal- |y, subcutaneously, or the like. Chimeric antibodies (mouse human hybrids) made by genetic engineering are also contemplated by this invention. Polyclonal antibodies are prepared by hyper-immunization of the animal with antigen. Then the blood of the animal is collected shortly after the repeated immunizations and the gamma globulin is isolated. Suitable methods for preparing polyclonal antibodies are described in the Handbook of Experimental Immunology, 3d edition, (ed. Weir, 1978). To obtain monoclonal antibodies, spleen cells from the immunized vertebrate demonstrating the desired antibody response are immortalized. The manner of immortalization is not critical, but the most common method is fusion with a myeloma fusion partner. Other techniques of immortaliza¬ tion include EBV transformation, transformation with bare DNA, such as oncogenes or retroviruses, or any other method which provides for stable maintenance of the cell line and production of monoclonal antibodies. The general process for obtaining monoclonal antibodies is described by Kohler and Milstein, in Nature. 256. 495-497 (1975), which is herein incorporated by reference. Human monoclonal antibodies may be obtained by fusion of the spleen cells with an appropriate human fusion partner, such as WI-L2, described in European Application No. 82.301103.6, the relevant portions of which are hereby incorporated by reference. A detailed technique for producing mouse x mouse monoclonal antibodies is taught by Oi and Herzenberg, in Selected Methods in Cellular Immunology. 351-372 (eds. Mishell and Shiigi, 1980), which also is herein incorporated by reference. The resulting hybridomas are screened to isolate individual clones, each of which secretes a single antibody species capable of recognizing the antigen. The peptides and/or antibodies may be used without modification or may be modified in a variety of ways, for example, by labeling. Labeling is intended to mean joining, either covalently or non-covalently, a moiety which directly or indirectly provides for a means of detection. A wide variety of labels are known and include: radionuclides, enzymes, substrates, cofactors, inhibitors, fluorescers, chemiluminescers, magnetic particles and the like. Many of the techniques for linking the peptides to suitable labels involve the use of their carboxyl groups, through the use of carbodiimide or active esters to form peptide bonds; the formation of sulfides by reaction of a mercapto group with an activated halogen, such as chloromethyl- styrene or activated olefin, such as maleimide; formation of a secondary amine by reaction of an amino group with a dialdehyde such as glutar- aldehyde, or the like, followed by reduction with sodium cyanoborohydride, or the like. An amino group-protected amino acid is bound in sequence through condensation of its reactive carboxyl group to the terminal amino group of the growing chain. After synthesizing the complete sequence, the peptide is removed from the insoluble carrier to yield free the peptide. This solid- phase approach is generally described by Merrifield, et al. in J. Am. Chem. Soc, 85, 2149-2156 (1963). The peptides of this invention can also be prepared through DNA techniques. The amino acid sequence of the desired peptide is used to deduce the codon sequence for the single-stranded DNA, synthesized using conventional synthetic techniques (including multiple gene copy techniques), then the double-stranded DNA is prepared and inserted at a suitable site in a cloning vehicle, vector, or plasmid. An appropriate organism, such as bacteria cells, yeast cells, or mammalian cells, is trans¬ formed to obtain expression of the desired peptide. The prepared peptides of the present invention can be isolated and purified from the reaction mixture by means of peptide separation, for example, by extraction, counter-current distribution, column chromato- graphy, high performance liquid chromatography, and the like. Brief Description of the Drawings Various other objects, features and attendant advantages of the present invention will be more fully appreciated as the same becomes better understood when considered in conjunction with the accompanying drawings, in which like reference characters designate the same or similar parts throughout the several views. Figure 1. Salmolysin Sequence. The sequence of the sly gene. The initiation codon is underlined and the termination codon is denoted by * . Sequences surrounded by the two boxes are Sspl sites used to construct ρSL2145. The DNA sequence is given as [Sequ.ID.No. 1] and the corresponding protein sequence of Salmolysin as [Sequ.ID.No. 2]. Primers used for PCR reactions are shown (Oligo 1-3; [Sequ.lD.No's. 3- 5]). Figure 2. Conservation of the sly Gene. Chromosomal DNA from clinical isolates of Salmonella was isolated, restricted with Pstl transferred to a Nytran, and probed with the labeled PCR amplified slyA fragment. A single 7 kb fragment hybridized to the probe with the exception of S. worthington, S. infantis, and S. enteritidis. The slyA gene may be multi¬ copy in these serotypes. A 5 kb fragment hybridizes with S. hadar. These data are summarized in Table 1. In the foregoing and in the following examples, all temperatures are set forth uncorrected in degrees Celsius and unless otherwise indicated, all parts and percentages are by weight. The entire disclosures of all applications, patents and publications, cited above, are hereby incorporated by reference. The pre-print entitled "Cytolytic Toxin Encoded by Salmonella is Required for Virulence and Survival within Macrophages" by Stephen J. Libby, Werner Goebel, Albrecht Ludwig, Nancy Buchmeier, Ferric C. Fang, Donald G. Guiney, Frances Bowe, J. Glenn Songer, and Fred Heffron, is attached to US Patent Application Ser. No. 08/054,480 as Appendix A, is hereby incorpored by reference. EXAMPLES Example 1: CLONING THE SALMOLYSIN GENE Although Salmonella is not usually hemolytic on laboratory culture media, weak zones of hemolysis around some colonies, especially in clinical isolates were occasionally observed. It was suspected that this variable phenomenon might be highly regulated and the putative hemolysin might only be expressed in vivo. Therefore, it was attempted to clone the cognate gene away from its normal regulatory elements. A cosmid clone bank was made from S. typhimurium ATCC 14028 in pl_AFR2, packaged into bacteriophage lambda in vitro, introduced by infection into E. coli strain LE392, and screened for hemolytic activity on blood agar plates. A detailed restriction map of a representative hemolytic clone was prepared and a series of deletions constructed to identify the location of the gene encoding the hemolysin. It was found that a 1.4 kb C/a//EcoRV fragment (ρSL1117) of the original 25 kb cosmid clone was required for hemolotic activity in E. coli. The nucleotide sequence of the 1.4 kb fragment was determined and analysis of the sequence adjacent to the Cial site revealed a 436 base open reading frame that could encode a protein of 16,747 daltons. The polymerase chain reaction (PCR), see, e.g., Innis et al., (1990), was used to amplify a 680 base pair region from chromosomal DNA encompassing the open reading frame. The sequence of this region is shown in Figure 1 [Sequ.ID.No. 1]. The PCR derived fragment was cloned into pKS (Stratagene, La Jolla, CA) and screened for hemolytic activity. This clone, pSL2070, was equally hemolytic as pSL1117, indicating that the 680 base-pair fragment contained all the genetic information necessary for the hemolytic phenotype in E. coli. The deduced protein sequence was used to search a translated version of the Genbank DNA database (updated August 1992). The closest matches were compared with slyA using the Fustell protein comparison matrix but no significant short homologies were found. Surprisingly, analysis of the salmolysin coding sequence did not reveal a classical signal sequence. However, it is still possible that salmolysin is exported to the outer membrane or extracellularly in vivo by a sec-independent pathway similar to export of the HlyA [R. A. Welch. Mol. Micro. Vol. 5, pp. 521-528 (1991 ) and Braun, V. & T. Focareta, Critical Reviews in Microbiology, Vol. 18, pp. 115-158 (1991 )]. Thus, a single gene, which we call s/yA for salmolysin, is sufficient to confer hemolytic activity on laboratory strains of E. coli. EXAMPLE 2. MAPPING AND CONSERVATION OF THE SALMOLYSIN GENE IN SALMONELLA sp. Since the map location for many Salmonella genes have been deter¬ mined, it was decided to map slyA to determine whether it corresponds to a genetically mapped but un-sequenced gene. The precise genetic location of the slyA gene was determined using a modification of the procedure described in [N.R. Benson and B.S. Goldman, J. Bact., Vol. 175, pp. 1673-1681 (1992) and P. Youdenan et al. Genetics, Vol. 118, pp. 581-592 (1988)]. Using this procedure, the slyA gene was mapped to a region between 28.5 and 30 minutes. Few genes have been identified in this location, either in Salmonella or in the related region in E coli [F.C. Neidhard, Escherichia coli and Salmonella typhimurium, Cellular and Molecular Biology, (American Society of Microboloby, Washington, D.C. (1987)]. DNA was extracted from the parent strain, 17 clinical Salmonella isolates, three Shigella serotypes, enteroinvasive E. coli (EIEC), and other pathogenic bacteria [ Materials and Methods for list). Southern hybridi- zation using the 680 bp fragment as a probe showed that the gene is present in all serotypes of Salmonella examined. Shigella and entero¬ invasive E. coli (EIEC) (Figure 2) but not in any of the other 25 bacterial species examined (data not shown). A 7 kb fragment hybridized in most serotypes of Salmonella. Three serotypes showed two fragments that hybridized, suggesting that the slyA gene may be duplicated in some sero¬ types of Salmonella or there are several Pstl sites in or around the slyA gene. The following Table 1 summarizes these results for the genus Salmonella. TABLE 1 : Sample Fragment 1.5 Kbp 5 Kbp 7 Kbp Salmonella typhimurium 14028a - (+) + typhimurium (clinical) + typhimurium (clinical) + heidelberg + montevideo + infantis - - + * ) choleraesuis + - + newport + hadar + enteritidis - - + * ) havana + barta + typhi + typhi 21A - - + worthington + - + cerro + arizona + typhisuis + Explanations: + band with given length of fragment (1.5, 5, 7 Kbp) (+) faint band with given length of fragment (1.5, 5, 7 Kbp) no band with given length of fragment (1.5, 5, 7 Kbp) * ) multiple bands around 7 Kbp EXAMPLE 3: BIOLOGICAL PROPERTIES OF SALMOLYSIN The size of the sly gene product was determined. SlyA was over- expressed by placing the coding region under the control of T7 RNA poly¬ merase using the PCR primers shown in Figure 1. A 519 bp fragment was amplified, cloned into pRK172 and transformed into E. coli BL21 DE [F.W. Studier et al., Methods in Enzvmology, Vol. 185, pp. 6-89 (1990)]. Proteins were labeled in the presence of IPTG (which induces expression of T7 RNA polymerase) and 35 S-methionine, then separated by SCS- PAGE. A single 16,000 dalton protein was specifically labeled. These results are in close agreement with the size of salmolysin predicted from the DNA sequence. Isolation of functional salmolysin was facilitated by the discovery that the hemolytic activity of the E. coli clones expressing the salmolysin gene could be released by osmotic shock. Overnight cultures of E. coli expres- sing cloned salmolysin (pSL1117) were osmotically shocked [F.W. Studier et al., Methods in Enzvmology. Vol. 185, pp. 6-89 (1990)]. Released salmolysin was partially purified by FPLC MonoQ anion exchange chroma- tography (Pharmacia); all hemolytic activity eluted at 50 mM NaCI. A 16,000 dalton protein, identical in size to the protein over-expressed under the control of T7 RNA polymerase, was enriched in this fraction. Identical¬ ly processed osmotic shock supematants of E. coli with pKS alone had no protein in this size range that eluted at 150 mM NaCI, nor was hemolysin activity detected in any fraction. Salmolysin was found to have an iso- electric point of pi 5.5 determined by preparative isoelectric focusing. Some of the biochemical properties of salmolysin were determined. The hemolytic activity of salmolysin was found to be sensitive to heat (65°C for 15 minutes) and was fully active in the presence of 50 mM EDTA, unlike the hemolysin from uropathogenic E. coli [ F.C. Neidhard, Escherichia coli and Salmonella typhimurium, Cellular and Molecular Biology, (American Society of Microboloby, Washington, D.C. (1987)]. No phospholipase or cholesterol oxidase activity was detected. Salmolysin is also able to lyse nucleated cells as well as a variety of erythrocytes (data not shown). Based on these results, a salmolysin is called a cytolysin. The preceding examples can be repeated with similar success by substituting the generically or specifically described reactants and/or operating conditions of this invention for those used in the preceding examples. From the foregoing description, one skilled in the art can easily ascertain the essential characteristics of this invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various usages and conditions. (1) GENERAL INFORMATION: (i) APPLICANT: (A) NAME: Merck Patent GmbH (B) STREET: Frankfurter Str. 250 (C) CITY: Darmstadt (E) COUNTRY: Germany (F) POSTAL CODE (ZIP): 64271 (G) TELEPHONE: 06151-727840 (H) TELEFAX: 06151-727191 (I) TELEX: 4 19 328 32 em d (ii) TITLE OF INVENTION: Cytolysin Gene and its Use for the Detection of Enterobacteriaceae (iii) NUMBER OF SEQUENCES: 5 (iv) COMPUTER READABLE FORM: (A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS (D) SOFTWARE: Patentln Release 81.0, Version 81.25 (EPO) (v) CURRENT APPLICATION DATA: APPLICATION NUMBER: WO (vi) PRIOR APPLICATION DATA: (A) APPLICATION NUMBER: US 08/054,480 (B) FILING DATE: 30-APR-1993 (vi) PRIOR APPLICATION DATA: (A) APPLICATION NUMBER: US 08/054,395 (B) FILING DATE: 28-APR-1993 (2) INFORMATION FOR SEQ ID NO:1: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 720 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: Salmonella (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: ATCGATGCTT TAGTTTTAGC CAAAACTGAA GCTACAGGTG CCAAGTGCGC ACTATGTCTG 60 AAAAAATGTC TATTGGTAAG CAAATTTAGC AATACATTTG TTTTGAGAAT ACAAATACTG 120 CACACTATTC TAAAATCAGC ATAATAACTT AGCAAGCTAA TTATAAGGAC. ATGAAATTGG 180 AATCGCCACT AGGTTCTGAT CTGGCACGGT TGGTGCGCAT TTGGCGTGCT CTGATTGACC 240 ATCGCCTCAA GCCTCTGGAA TTGACGCAGA CACATTGGGT CACGTTGCAC AATATTCATC 300 AATTGCCGCC TGACCAGTCG CAGATTCAAT TGGCTAAAGC GATAGGCATT GAGCAGCCAT 360 CGCTGGTACG CACGTTGGAT CAACTTGAAG ATAAGGGGCT AATTTCGCGG CAAACCTGCG 420 CCAGCGATCG TCGCGCTAAG CGGATTAAAC TGACCGAAAA AGCGGAGCCG CTGATCGCTG 480 AGATGGAAGA GGTCATTCAT AAAACGCGCG GTGAAATTTT GGCTGGGATT TCTTCAGAGG 540 AGATTGAGCT TCTGATTAAA CTTATCGCCA AACTTGAACA CAATATTATG GAATTGCACT 600 CTCACGATTG AGGTGCAGGG GCATACGTGT GGCCATGTGA CCACACGTAA AGCCTGGTTT 660 AGCGTGGAGA GACGGTAACC TGGCTGCCGT TGCTGGCCAG CACGACACGC TGACCTGCCG 720 (2) INFORMATION FOR SEQ ID NO:2: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 146 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: Salmonella (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: Met Lys Leu Glu Ser Pro Leu Gly Ser Asp Leu Ala Arg Leu Val Arg 1 5 10 15 lie Trp Arg Ala Leu lie Asp His Arg Leu Lys Pro Leu Glu Leu Thr 20 25 30 Gin Thr His Trp Val Thr Leu His Asn lie His Gin Leu Pro Pro Asp 35 40 45 Gin Ser Gin He Gin Leu Ala Lys Ala lie Gly He Glu Gin Pro Ser 50 55 60 Leu Val Arg Thr Leu Asp Gin Leu Glu Asp Lys Gly Leu He Ser Arg 65 70 75 80 Gin Thr Cys Ala Ser Asp Arg Arg Ala Lys Arg lie Lys Leu Thr Glu 85 90 95 Lys Ala Glu Pro Leu lie Ala Glu Met Glu Glu Val He His Lys Thr 100 105 ' 10 Arg Gly Phe He Leu Ala Gly He Ser Ser Glu Glu He Phe Leu Leu 115 120 125 He Lys Leu He Ala Lys Leu Glu His Asn He Met Glu Leu His Ser 130 135 140 His Asp 145 (2) INFORMATION FOR SEQ ID NO:3: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 23 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: Salmonella (xi) SEQUENCE DESCRIPTION: SEQ ID N0:3: ATCGATGCTT TAGTTTTAGC CAA 23 (2) INFORMATION FOR SEQ ID NO:4: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 28 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: Salmonella (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: ATTATAAGGA GATGAAATTG GAATCGCC 28 (2) INFORMATION FOR SEQ ID NO:5: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 26 base pairs (B) TYPE: nucleic acid (C) STRA.viDED ESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: Salmonella (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5: TTAGCGTGGA GAGACGGTAA CCTGGC 26

Claims: CLAIMS 1. A DNA having a DNA sequence according to SEQU.ID.NO.1. 2. A DNA of claim 1 , coding the protein of SEQU.ID.NO.2 and representing a partial sequence starting at position 171 and ending at position 609 of SEQU.ID.NO.1. 3. An isolated gene comprising a DNA of claim 1 or 2. 4. An isolated nucleic acid obtained from a naturally-occurring source which hybridizes to the DNA of claim 1 or 2. 5. An oligonucleotide comprising a DNA sequence which is specific for the DNA sequence of claim 1 or 2. 6. The oligonucleotide of claim 5, having a sequence according to SEQU.ID.NO.3, SEQU.ID.NO.4, or SEQU.ID.NO.5. 7. A method of detecting the presence of the sly A gene, comprising hybridizing a nucleic acid specific for the slyA gene with a nucleic acid containing sample in which the presence of the slyA gene is to be detected, the conditions of such hybridizing being effective for said specificity of the slyA gene; and detecting the presence or absence of binding between the nucleic acid specific for slyA and the nucleic acid of the sample. 8. The method of claim 7, wherein the sample is a food stuff. 9. The method of claim 7, wherein the sample is blood, serum, plasma, urine or feces. 10. A method of detecting the presence of the sly A gene, comprising hybridizing two different oligonucleotides according to claim 5 with nucleic acid containing sample in which the presence of the slyA is to be detected, the conditions of such hybridizing being effective for said specificity of the slyA gene; performing the polymerase chain reaction on the hybridized sample; and detecting the presence or absence of a DNA fragment formed by the polymerase chain reaction. 11. A kit for detecting the presence of an organism expressing a slyA gene comprising a container having one or more components necessary for detection of the gene and another container having an oligonucleotide according to claim 5. 12. A pair of single-stranded nucleic acid primers for amplification of a region of DNA comprising a slyA gene.

Inventors: GOEBEL WERNER, HEFFRON FRED L, LIBBY STEPHEN J, GOEBEL, WERNER, HEFFRON, FRED, L., LIBBY, STEPHEN, J.
Applicants: MERCK PATENT GMBH
Application year: 1994
Main classification: C12N15
IPC codes: C07H21, C07K14, C12N15, C12Q1, G01N33